Lee Soon-Tae, Chu Kon, Kim Eun-Hee, Jung Keun-Hwa, Lee Kyung-Bok, Sinn Dong-In, Kim Seung U, Kim Manho, Roh Jae-Kyu
Stroke and Neural Stem Cell Laboratory in Clinical Research Institute, Stem Cell Research Center, Department of Neurology, Seoul National University Hospital, Seoul, South Korea.
J Neurosci Methods. 2006 Oct 30;157(2):225-9. doi: 10.1016/j.jneumeth.2006.04.019. Epub 2006 Jun 2.
Few sensitive and reliable methods have been available for quantifying the number of transplanted human neural stem cells (hNSC) in the animal brain. To develop an accurate method for quantifying the number of hNSC incorporated in rat brain, we performed real-time PCR on hNSC-transplanted rat brains using a target sequence for ERV-3, which is an endogenous retrovirus present with a known copy number in all human cells, but not present in rodent cells. A standard curve was developed for known amount of different mixes of hNSC and rat fibroblasts, and test samples were prepared by manually incorporating variable, predefined numbers of hNSCs into rat brains. A cerebral rat hemisphere injected with 10(7) hNSC revealed 1.125% chimerism. Moreover, a linear correlation was found between hNSC numbers injected and their concentrations in the rat brain. In conclusion, the developed quantitative ERV-3 assay enables a simple, fast, and reproducible detection and quantitation of hNSC numbers in the rat brain.
几乎没有灵敏且可靠的方法可用于定量动物脑中移植的人类神经干细胞（hNSC）的数量。为了开发一种准确的方法来定量大鼠脑中整合的hNSC数量，我们使用ERV-3的靶序列对hNSC移植的大鼠脑进行了实时PCR，ERV-3是一种内源性逆转录病毒，在所有人类细胞中以已知拷贝数存在，但在啮齿动物细胞中不存在。针对已知数量的不同hNSC和大鼠成纤维细胞混合物绘制了标准曲线，并通过将预先定义的可变数量的hNSC手动注入大鼠脑中来制备测试样品。注射了10(7)个hNSC的大鼠脑半球显示出1.125%的嵌合率。此外，在注射的hNSC数量与其在大鼠脑中的浓度之间发现了线性相关性。总之，所开发的定量ERV-3检测方法能够简单、快速且可重复地检测和定量大鼠脑中的hNSC数量。
